Comprehensive circulating microRNA profile as a supersensitive biomarker for early-stage lung cancer screening.

PURPOSE:  Less-invasive early diagnosis of lung cancer is essential for improving patient survival rates. The purpose of this study is to demonstrate that serum comprehensive miRNA profile is high sensitive biomarker to early-stage lung cancer in direct comparison to the conventional blood biomarker using next-generation sequencing (NGS) technology combined with automated machine learning (AutoML). METHODS: We first evaluated the reproducibility of our measurement system using Pearson's correlation coefficients between samples derived from a single pooled RNA sample. To generate comprehensive miRNA profile, we performed NGS analysis of miRNAs in 262 serum samples. Among the discovery set (57 patients with lung cancer and 57 healthy controls), 1123 miRNA-based diagnostic models for lung cancer detection were constructed and screened using AutoML technology. The diagnostic faculty of the best performance model was evaluated by inspecting the validation samples (74 patients with lung cancer and 74 healthy controls). RESULTS: The Pearson's correlation coefficients between samples derived from the pooled RNA sample ≥ 0.98. In the validation analysis, the best model showed a high AUC score (0.98) and a high sensitivity for early stage lung cancer (85.7%, n = 28). Furthermore, in comparison to carcinoembryonic antigen (CEA), a conventional blood biomarker for adenocarcinoma, the miRNA-based model showed higher sensitivity for early-stage lung adenocarcinoma (CEA, 27.8%, n = 18; miRNA-based model, 77.8%, n = 18). CONCLUSION: The miRNA-based diagnostic model showed a high sensitivity for lung cancer, including early-stage disease. Our study provides the experimental evidence that serum comprehensive miRNA profile can be a highly sensitive blood biomarker for early-stage lung cancer.

Efficacy of endobronchial ultrasound-guided transbronchial biopsy without guide sheath for small peripheral pulmonary lesions (≤15 mm): A retrospective cohort study.

INTRODUCTION: Endobronchial ultrasonography-guided transbronchial biopsy (EBUS-TBB) with guide sheath (GS) is an effective procedure for diagnosing small peripheral pulmonary lesions (PPLs) (≤20 mm in the largest diameter). However, samples obtained using EBUS-TBB with GS are small, and the diagnostic yield of small PPLs biopsied using EBUS-TBB with GS is unsatisfactory. OBJECTIVES: The aim of this study was to evaluate the diagnostic yield of small PPLs using EBUS-TBB without GS compared to that with GS. MATERIALS AND METHODS: Between 1 April 2013 and 31 March 2015, 276 consecutive lesions were biopsied using EBUS-TBB with GS or without GS. We retrospectively compared EBUS-TBB with and without GS in terms of the diagnostic yield and complications related to small PPLs (≤20 mm). RESULTS: Of the 276 lesions who underwent EBUS-TBB with or without GS, we identified 80 lesions with small PPLs (≤20 mm). Sixty-two lesions were successfully diagnosed by EBUS-TBB (77.5%, diagnostic yield). The diagnostic yield of PPLs using EBUS-TBB without GS was not significantly higher than that using EBUS-TBB with GS (34/41 = 82.9% and 28/39 = 71.7%, respectively; p = 0.233). However, according to size (≤15 mm or > 15 mm), location (upper, middle/lingular, or lower area), and structure (solid nodule or ground-glass opacity), the diagnostic yield of small PPLs (≤15 mm) using EBUS-TBB without GS was significantly higher than with GS (21/26 = 80.7% vs. 8/16 = 50.0%, p = 0.036). There were no complications among the two groups. CONCLUSIONS: EBUS-TBB without GS is an effective and safe procedure for diagnosing small PPLs (≤15 mm) compared to that with GS.

Hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

BACKGROUND: Persistent hypoxia stimulation, one of the most critical microenvironmental factors, accelerates the acquisition of epithelial-mesenchymal transition (EMT) phenotypes in lung cancer cells. Loss of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression might accelerate the development of lung cancer in vivo. Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes. Nevertheless, it is not known whether persistent hypoxia can modulate PTEN phosphatase activity or whether hypoxia-induced EMT phenotypes are negatively regulated by the PTEN phosphatase activity. We aimed to investigate hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers. METHODS: Western blotting was performed in five lung cancer cell lines to evaluate total PTEN expression levels and the PTEN activation. In a xenograft model of lung cancer cells with endogenous PTEN expression, the PTEN expression was evaluated by immunohistochemistry. To examine the effect of hypoxia on phenotypic alterations in lung cancer cells in vitro, the cells were cultured under hypoxia. The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system. RESULTS: Lung cancer cells involving the EMT phenotypes showed a decrease in total PTEN expression and an increase in p-PTEN. In a xenograft model, loss of PTEN expression was observed in the tumor lesions showing tissue hypoxia. Persistent hypoxia yielded an approximately eight-fold increase in the p-PTEN/PTEN ratio in vitro. PTEN4A did not affect stabilization of hypoxia-inducible factor 1α. PTEN4A blunted hypoxia-induced EMT via inhibition of ß-catenin translocation into the cytoplasm and nucleus. CONCLUSION: Our study strengthens the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancer cells under persistent hypoxia.

Involvement of TGFß-induced phosphorylation of the PTEN C-terminus on TGFß-induced acquisition of malignant phenotypes in lung cancer cells.

Transforming growth factor ß (TGFß) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFß-induced translocation of ß-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via ß-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFß can modulate both ß-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFß-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFß stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFß-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of ß-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFß-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFß-induced malignant phenotypes in lung cancer cells.

Differential modulation of surfactant protein D under acute and persistent hypoxia in acute lung injury.

Hypoxia contributes to the development of fibrosis with epithelial-mesenchymal transition (EMT) via stimulation of hypoxia-inducible factor 1α (HIF-1α) and de novo twist expression. Although hypoxemia is associated with increasing levels of surfactant protein D (SP-D) in acute lung injury (ALI), the longitudinal effects of hypoxia on SP-D expression in lung tissue injury/fibrosis have not been fully evaluated. Here, the involvement of hypoxia and SP-D modulation was evaluated in a model of bleomycin-induced lung injury. We also investigated the molecular mechanisms by which hypoxia might modulate SP-D expression in alveolar cells, by using a doxycycline (Dox)-dependent HIF-1α expression system. Tissue hypoxia and altered SP-D levels were present in bleomycin-induced fibrotic lesions. Acute hypoxia induced SP-D expression, supported by the finding that Dox-induced expression of HIF-1α increased SP-D expression. In contrast, persistent hypoxia repressed SP-D expression coupled with an EMT phenotype and twist expression. Long-term expression of HIF-1α caused SP-D repression with twist expression. Ectopic twist expression repressed SP-D expression. The longitudinal observation of hypoxia and SP-D levels in ALI in vivo was supported by the finding that HIF-1α expression stabilized by acute hypoxia induced increasing SP-D expression in alveolar cells, whereas persistent hypoxia induced de novo twist expression in these cells, causing repression of SP-D and acquisition of an EMT phenotype. Thus this is the first study to demonstrate the molecular mechanisms, in which SP-D expression under acute and persistent hypoxia in acute lung injury might be differentially modulated by stabilized HIF-1α expression and de novo twist expression.

Involvement of the transcription factor twist in phenotype alteration through epithelial-mesenchymal transition in lung cancer cells.

Epithelial-mesenchymal transition (EMT), which involves the persistent loss of epithelial markers and expression of mesenchymal markers, is assumed to have a critical role in not only tissue development during embryogenesis but also central mechanisms that enhance the invasive and metastatic ability of cancer cells. Twist has been identified to play an essential role in EMT-mediated tumor invasion and metastasis. Although recent studies suggest that twist expression levels in tissue specimens of lung cancer might be associated with prognosis, the expression of twist in lung cancer cells itself and its effect have not been fully evaluated. Here, we evaluated twist expression and its effect on phenotype alteration in lung cancer cell lines. Twist expression varied among human lung cancer cell lines. The lung cancer cell lines with high twist expression also tended to show a high vimentin/E-cadherin ratio, which was supported by a migration assay, in which high twist expression gave rise to high cell motility. Furthermore, in comparison to control cells, the lung cancer cells with ectopic expression of twist showed a significant phenotype alteration through EMT and an increasing ability to migrate in vitro, in part, due to a tenfold increase in matrix metalloproteinases activity and almost a 60% increase in modulation of focal adhesion kinase activity, although a contribution of microRNA appeared unlikely in our study. Our present analysis of twist expression in lung cancer provide clues to comprehensive understanding of the mechanisms, by which metastasis often develops in lung cancer.